Validation of Housekeeping Genes for Gene Expression Analysis in Iwagaki Oyster ( Crassostrea nippona ) Under Salinity S
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Validation of Housekeeping Genes for Gene Expression Analysis in Iwagaki Oyster (Crassostrea nippona) Under Salinity Stress by Quantitative Real-Time PCR GONG Jianwen1), LI Qi1), 2), *, YU Hong1), LIU Shikai1), and KONG Lingfeng1) 1) Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao 266003, China 2) Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China (Received December 26, 2019; revised February 13, 2020; accepted June 30, 2020) © Ocean University of China, Science Press and Springer-Verlag GmbH Germany 2020 Abstract Hypo-salinity can reduce the immunological reaction in Crassostrea nippona, even lead to massive mortality. It is important to understand the molecular mechanism of oyster defense system, while quantitative real-time PCR can be employed in the study. However, the accuracy of quantitative real-time PCR relies on the use of suitable reference genes. In this study, the expression stability of 14 candidate reference genes including traditional housekeeping genes EF1A, TUB, TUA, GAPDH, RO21, as well as new candidate reference genes RPL5, RPL8, RPS27, RPL14, RPL4, CO3, RPS8, RPS4, CYTB in different tissues of C. nippona under salinity stress has been validated by quantitative real-time PCR. Ribosomal protein genes selected through expression analysis of transcriptome data from C. nippona generally were more stable than traditional reference genes. According to the geNorm analysis, RPL4 and RPS4 could be used as internal controls for studying gene expression in C. nippona with real-time PCR under salinity stress. Key words
Crassostrea nippona; reference gene; hypo-salinity stress; ribosomal protein genes
1 Introduction Quantitative real-time PCR (qRT-PCR) has been widely used to measure gene expression because of its high sensitivity, flexibility, and reproducibility (Heid et al., 1996). The variations caused by differences in samples, RNA extraction, efficiency of enzyme and transcriptional activity can influence the experimental accuracy (Mackay et al., 2002). For accurate and reliable analysis of target gene expression, normalization of qRT-PCR data with suitable internal reference gene(s) is required (Radonić et al., 2004). An ideal reference gene should express at stable level in all tissues, regardless of the experimental conditions or treatments (Vandesompele et al., 2002; Radonić et al., 2004). Commonly used reference genes usually are those involved in basic cellular processes, such as the components of cytoskeleton, glycolytic pathway, protein folding and degradation (Eisenberg and Levanon, 2003). However, evidences show that transcription levels of housekeeping genes vary considerably in different tissues and under variable conditions (Greer et al., 2010). Therefore, selecting mul* Corresponding author. Tel: 0086-532-82031622 E-mail: [email protected]
tiple stably expressed reference genes, other than the commonly used housekeeping genes, is important for
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